Sample component trapping, release, and separation with membrane assemblies interfaced to electrospray mass spectrometry

ABSTRACT

A method and apparatus to trap, release and/or separate sample components in solution passing through a channel with or without packing material present by passing ion current through the channel driven by an electric field. A portion of the ion current includes cation and/or anion species generated from second solution flows separated from the sample solution flow path by semipermeable membranes. Cation and/or anion ion species generated in the second solution flow regions are transferred into the sample solution flow path through ion selective semipermeable membranes. Ion current moving along the sample solution flow path is controlled by varying the composition of the second solutions and/or changing the voltage between membrane sections for a given sample solution composition. The sample composition may also be varied separately or in parallel to enhance trapping, release and/or separation efficiency and range.

RELATED PATENTS AND PATENT APPLICATIONS

This application claims the priority of U.S. Provisional PatentApplication Ser. No. 60/840,095, filed on Aug. 25, 2006, and U.S. patentapplication Ser. No. 11/132,953 both of which are incorporated herein byreference. U.S. Pat. No. 4,542,293 is also incorporated herein byreference.

FIELD OF INVENTION

This invention relates to the field of separation of sample componentsusing electric fields and ion currents in solution with remote cationand/or anion species generation in solution flows off-line with non MassSpectrometer detectors or integrated on-line with ElectrosprayIonization or other Atmospheric Pressure Ion Source interfaced to a MassSpectrometer.

BACKGROUND

The invention includes apparatus and methods that enables or enhancescapture, release and/or separation of analyte compounds in a samplesolution using electric fields and ion current flow through the samplesolution channel with cation and/or anion exchange through semipermeablemembranes with multiple second solution flow paths. Cations or anionsgenerated or present in the sample solution or in one or more secondsolution flow paths are transferred through ion selective semipermeablemembranes into or out of the sample solution flow channel to effecttrapping, electrocapture, binding, displacement, release and/orisoelectric focusing of sample components in the sample solution. Theinvention comprises a stand alone separation method and apparatus usingnon mass spectrometer detectors or may be connected to or integratedwith an Electrospray Ionization or other Atmospheric Pressure ion sourceinterfaced to a Mass Spectrometer analyzer. The invention apparatus andmethods may be scaled to accommodate higher or lower sample solutionliquid flow rates.

The invention may be configured in a high pressure liquid chromatography(HPLC) apparatus and methods with packed columns or can be applied tomethods and apparatus employing low pressure packed or open tubularcolumn sample separation techniques. The invention comprises one or moresemipermeable membrane assemblies positioned along a sample solutionflow channel with membrane assemblies separating a sample bearing firstsolution from one or more second solution solution flows. Each secondsolution flow can have a different composition and each second solutioncomposition can change over time using solution composition gradients orstep functions. Packed or open channels, where sample componentseparation occurs, may be configured in a membrane assembly, betweenmembrane assemblies or positioned upstream or downstream of membraneassemblies. Ions generated in the second solution flow paths aretransferred through the semipermeable membrane into the sample solutionflow driven by the applied electric field. Conversely, ions in thesample solution can be transferred into a second solution through thesemipermeable membranes. Ions can be selectively added to or removedfrom the sample solution flow path at one or more membrane sectionspositioned along the sample solution flow path. The ion current passingthrough the semipermeable membrane into the sample solution issubsequently driven along the length of the sample solution channel by avoltage gradient maintained along a portion of the sample solution flowchannel length. Individually controlled voltages are applied toelectrodes in contact with each second solution flow. Multiplesemipermeable membrane sections positioned along the sample solutionflow path allow the application of different electric fields and ioncurrents at different locations in the sample solution flow path. Ionsentering the sample solution through one membrane can be remove throughan adjacent membrane along the sample solution flow path forming localtrapping, capture, release or separation regions.

Configuring one or more semipermeable membrane sections withElectrospray ionization allows independent control and optimization ofthe capture, trapping, release and/or separation of sample species andElectrospray ionization. Membrane materials and second solutioncompositions may also be configured to allow selected neutral species totraverse a membrane from a second solution into the sample solution orconversely from the sample solution into a second solution. The transferof neutral species through a semipermeable membrane is driven orcontrolled by controlling the relative concentration of the neutralspecies of interest across each membrane. The added ion or ion andneutral species selectively introduced into or removed from the samplebearing first solution changes the solution pH and/or solution chemistrycausing or enhancing binding or release of sample components andeffecting separation, cleanup or reactions of analyte species. Theseprocesses can also be used to simultaneously optimize or enhance theperformance if an Atmospheric Pressure Ion (API Source) such asElectrospray.

Separations of mixtures of analyte components in a solution is widelypracticed using packed Liquid Chromatography (LC) separations, open tubeCapillary Electrophoresis (CE) and more recently open tubeElectrocapture (EC). Liquid Chromatography separation is effected bybinding or partial binding of an analyte to a solid phase or materialpacked within the chromatography column as a liquid or mobile phase flowpasses through the column. The liquid flow through the column can be runisocratically, having constant composition, or with changingcomposition, usually in the form of a gradient or a series of steps.When running isocratic liquid chromatography, analytes are separated inthe flowing solution by size differences or differences in partialbinding energy with the surface chemistry or phase of material packedwithin the liquid chromatography column volume. Analyte species thatexhibit stronger binding to the column phase will elute from the columnat a later time then those analytes with weaker binding energy. Analytecomponents eluting from a chromatography column are separated bothspatially in the solution flow and temporally. When gradient liquidchromatography separations are conducted, the chemistry of the solutionpassing through the column is varied in a controlled manner to releaseanalyte bound to the column phase material at specific times. Analyteswith different binding energies and/or different solution chemistryrelease conditions will be separated in solution flowing through theliquid chromatography column. Separation of analytes in solution occurdue to the partitioning of attractive and release forces on a givenanalyte species based on the differential interactions between thestationary and mobile phases in a liquid chromatography column.Attractive and release forces are manipulated by changing solutionpolarity, pH and ionic or buffer species concentration. Analytes areseparated in open tube Capillary Electrophoresis and Electrocapture by abalance of electric fields, ion mobility and in the case ofElectrocapture, convective solution flow.

The most commonly used types of high pressure liquid chromatographyinclude Reverse Phase (RP), Normal Phase (NP), Ion Exchange (IE) andSize Exclusion (SE) separations. None of these separation techniques arepracticed with an ion current passing through the LC column. CapillaryElectrophoresis (CE) employs an electric field maintained in the samplesolution along an open CE column length to effect separation of analytespecies through differential electroosmotic migration of analyte speciesthrough a solution with electroosmotic flow (EOF) along the CE columnlength. Variations in Capillary Electrophoresis have been developed,such as Capillary Electrochromatography (CEC), that combine useelectroosmotic separation of CE with the partitioning separation of LCdue to differential interactions between two phases. Typically in CE andCEC the sample solution composition remains constant throughout aseparation run. Semipermeable membranes have been configured by SeversJ. C., and Smith R. D., Anal. Chem. 1997, 69, 2154-2158, at the exit endof CE columns to complete the electrical circuit in a CE-ElectrosprayMass Spectrometer interface. Capillary electrophoresis separation ofanalyte species was used in this apparatus and method. No liquidchromatography separation was described using this interface and no ionspecies passing through the membrane flowed through the CE column toenhance or cause species separation in solution.

Electrocapture of sample components in solution has been employed tocapture sample components in an open column liquid flow stream withsubsequent release of components. Electrocapture or analyte species inan open liquid flow column or channel allows preconcentration of samplesprior to separation with CE, sample preparation such as desalting,reaction with reagent species and effecting separation of components inthe solution flow as described by Juan Astorga-Wells et. al., U.S.Patent Number US 2005/0284762 A1, Sag-Ryoul Park and Herold Swerdlow,Anal. Chem. 2003, 75, 4467-4474 and Juan Astorga-Wells, Hans Jornaval,and Tomas Bergman, Anal. Chem. 75, 5213-5219. Electrocapture of speciesis effected using a balance of electric fields, ion mobility andhydrodynamic flow along a liquid flow channel length. Semipermeablemembranes separating the sample solution flow from anode and cathodeelectrodes immersed in reservoirs containing static conductive solutionswith no flow have been configured in Electrocapture devices as describedin the above references. As described by the authors, sample componentsin solution have been captured with hydrodynamic forces balanced againstan electric field along the sample flow path or on a semipermeablemembrane. Release of components can be realized by changing the voltagebetween the anode and cathode, changing the sample solution flow rateand/or changing the sample solution composition. The Electrocapturedevices have no second solution flow to replenish charge in the anodeand cathode reservoirs. When the sample flow is stopped, the current inthe sample flow channel stops due to charge depletion. In the presentinvention, second solution flow supplies charged species to the samplesolution flow path through the semipermeable membranes and replenishesthe charge during operation. The present invention requires noelectrolytes added to the sample solution and the electrical currentmaintained along the sample solution flow path can be changed bymodifying the second solution composition with no need to changerelative electrode voltage or no requirement to change the compositionof the sample solution flow to effect capture and/or release of analytecomponents in the sample flow.

The use of semipermeable membranes to exchange unwanted ion species inan eluant flow eluting from a liquid chromatography column with adesired ion species has been described previously in EPA PublicationNumbers 32,770, 69,285 and 75,371, 69,285 and 180,321 with publicationdates Jul. 29, 1981, Jan. 12, 1983, Mar. 30, 1983 and May 7, 1986respectively. This technique generically described as ion suppression iswidely practiced in ion exchange chromatography (IEC) to reduce theconductivity of eluant exiting an IEC column prior to passing through aconductivity detector. Separation is often achieved in IEC by displacinganalyte bound to the column stationary phase by charge with a displacinganion or cation added to eluant flow. The anion or cation species,typically added as a net neutral salt, hydroxide or acid compound to theeluant flow passing through an IEC column, can be removed after the IECcolumn exit by charged species exchange through flat or cylindricalsemipermeable membranes as described in the above EPA publications. Theselective reduction of solution conductivity without the reduction ofanalyte species in IEC eluant flow improves the conductivity detectionlimits of analytes separated while passing through an IEC column. Dualmembrane devices are configured wherein the eluant flow exiting the IECcolumn is in contact with two semipermeable membranes which separate theeluant liquid from two second solutions flowing on the opposite side ofboth membranes. A voltage is applied between electrodes in contact withboth second solution flows driving charged species of one polarity fromone second solution flow into the eluant flow while simultaneouslydriving a charged species in the eluant flow through the second membraneinto the second eluant flow. This effectively exchanges charged speciesin a net neutral eluant flow between the IEC column exit and aconductivity detector. Multiple layer membrane assemblies have beenconfigured to provide exchange or suppression of charged species ineluant flow exiting an IEC column while regenerating the second solutionneutral salt, acid or base composition used to exchange ion species.

Similar semipermeable membrane devices have been configured to provideselected cation or anion species with counter ions in aqueous eluantflow as described in U.S. Pat. No. 5,045,204. Two or more membraneassemblies have been configured in contact with the eluant exiting froman IEC column providing the dual function of exchanging ion species toreduce conductivity while simultaneously adding the removed ion speciescombined with a counter ion to an aqueous solution. The aqueous solutionwith the neutral salt, acid or base species is used as the eluant flowentering the IEC column as described in U.S. Pat. No. 5,045,204. Theexchange of ion species, as described, occurs in an ion exchange resinbed configured downstream of the IEC column exit and after the region ofchromatography separation in an ion chromatography system. No ioncurrent passes through the separation or LC column or separation media.In the apparatus and methods described, a net electrically neutral fluidflow passes through the IEC column and no ion current passes through theIEC column. Gradients of ion species with counter ions can be generatedpost column in the IEC eluant flow exiting the IEC column by increasingthe ion current passing through the semipermeable membranes. This iseffected by changing second solution composition or the electricalpotential applied between electrodes in contact with the secondsolutions.

The present invention provides the addition and/or removal of chargedspecies through one or more semipermeable membranes to an eluant flowthrough a liquid chromatography column, an Ion Exchange column, a CEcolumn and/or an Electrocapture flow channel. The invention providesdirect ion current through an LC or IEC column or through an unpackedchannel. No counter ion is added to the eluant flow and the addition ofcharged species to the eluant flow through the semipermeable membranesmatches the electrical current passing through the LC or IEC column oropen sample solution channel length. Charged species added from onesecond solution into the sample solution eluant flow through a firstsemipermeable membrane can be removed by passing equal ion current fromthe sample solution through a second semipermeable membrane into adifferent second solution flow positioned at the opposite end of the LC,IEC column or open channel. The semipermeable membrane materials andsecond solution compositions can be selected to introduce chargedspecies and/or organic modifiers into the eluant flow from one or moresecond solution flows to effect or improve ion exchange, reverse phasechromatographic, CEC or Electrocapture separation of analyte species insolution.

In different embodiments of the invention one or more membraneassemblies are configured with an Electrospray (ES) ion source or otherAPI source interfaced to a mass spectrometer (MS). Such embodiments ofthe invention can be configured wherein sample separation can beperformed integrated with the Electrospray process or the sampleseparation process can be conducted and optimized independent fromElectrospray Mass Spectrometer (ES/MS) processes. Embodiments of theinvention provide an efficient and precise means of adding chargedspecies to an eluant flow to effect or enhance chromatographic orElectrocapture separation while simultaneously optimizing Electrosprayionization source mass spectrometer performance. Configuring anElectrospray ion source with a semipermeable membrane assembly wherebyElectrospray current is generated in a second solution flow andtransferred through the semipermeable membrane is described in U.S.Pending application Ser. No. 11/132,953 included herein by reference.

SUMMARY OF THE INVENTION

The present invention enables the controlled addition and removal of ionor neutral species into eluant flow without counter ions or the exchangeof ions of like charge in liquid chromatography, IEC or Electrocapturepacked or open solution channel analyte species separations. Ion orneutral species are transferred through one or more semipermeablemembranes between a sample solution and one or more second solutionflows to effect sample component capture, release and separation in thesample solution flow path while enabling optimization of Electrosprayionization and mass spectrometer performance. The invention can beconfigured with electrical current passing through the sample solutionflow channel packed with separation media or configured as open flowchannel. Cation or Anion species pass through the packed LC column oropen sample solution flow path as electrical current in directproportion to the electrical current passing through the semipermeablemembranes positioned upstream and downstream in the sample solutionflowpath. For a given sample solution composition, the ion current andion species passing through the packed or open sample solution flowchannel can be controlled by adjusting the voltage applied to electrodesin the second solution flowpaths of membrane assemblies and/or changingthe composition of the second solutions through gradients or stepfunctions. The total Electrospray current can be controlled independentof the direction or magnitude of the upstream ion current passing alongthe sample solution flow path. Single or multiple semipermeable membraneassemblies can be configured with one or more sections of packed and/oropen sections of sample solution flow path and with one or moredetectors including ultraviolet light absorption, conductivity,condensation nuclei and mass spectrometer detectors. The invention canbe configured with or integrated into an Electrospray ion source orother API source interfaced to a mass spectrometer. The independentcontrol of ion current, cation or anion species and sample solutioncomposition in the sample solution flow channel and into specific LC,IEC, CE or CEC column and detector types allows independent optimizationof sample component separation and detector performance.

The invention provides independent adjustment of the following variablesto optimize sample component trapping, release and/or separation insolution while independently optimizing Electrospray or other API sourceand Mass Spectrometer or other detector performance;

-   -   1. Second solution composition can be changed independently        using gradients and/or step functions in each membrane assembly        during a run.    -   2. Relative voltage amplitude and polarity applied to electrodes        in second solution flow paths of adjacent membrane assemblies        can be independently adjusted during a run.    -   3. Semipermeable membrane materials can be configured to pass        selected ion and/or neutral species.    -   4. The sample solution composition can be changed using a        gradient or a step function during a run.    -   5. The relative voltages applied to Electrospray ion source or        other API source electrodes can be changed independently of        upstream second solution electrode voltages.

Each voltage, and solution composition variable can be independentlycontrolled in a synchronized manner through manual or programmedsoftware control.

The invention comprises different combinations and configurations ofmembrane assemblies, packed sections and open sections of the samplesolution flow path, Electrospray probe assemblies configured with andwithout pneumatic nebulization assist and different detector typesincluding but not limited to mass spectrometry, UV absorption,conductivity and particle counting. Embodiments of the invention caninclude but are not limited to the following component combinations:

-   -   1. A single semipermeable Membrane assembly positioned upstream        of an Electrospray inlet probe with the Electrospray spray        sample solution flow tube comprising a packed LC column.    -   2. Two semipermeable Membrane assemblies configured at the        entrance and exit end of a packed LC column with an Electrospray        inlet probe positioned downstream of the exit end Membrane        assembly.    -   3. Three semipermeable Membrane assemblies with downstream        Membrane assembly one providing Electrospray ion current and a        packed LC column configured between the upstream Membrane        assemblies two and three configured in series along the sample        solution flow path.    -   4. Three semipermeable Membrane assemblies with the first        downstream Membrane assembly providing Electrospray ion current        and an open sample solution flow channel configured between        upstream Membrane assemblies two and three configured in series        along the sample solution flow path.    -   5. Three semipermeable Membrane assemblies with the first        downstream Membrane assembly providing Electrospray ion current,        a packed or open sample solution flow channel configured between        the second and third upstream Membrane assemblies and an        Ultraviolet light absorption detector cell positioned between        the first and second Membrane assembly in the sample solution        flow path.    -   6. Four semipermeable Membrane assemblies configured in the        sample solution flow path with the first down stream Membrane        assembly providing Electrospray ion current and with open        channel sections of the sample solution flow path configured        between upstream Membrane assemblies 2 and 3 and 3 and 4        respectively.    -   7. Four semipermeable Membrane assemblies configured in the        sample solution flow path with the first down stream Membrane        assembly providing Electrospray ion current and a packed LC        column configured between upstream Membrane assemblies three and        four and an open sample solution channel section configured        between upstream Membrane assemblies two and three.

The configuration and operation of each embodiment of the inventionlisted above may be varied. The Electrospray (ES) ion source interfacedto a mass spectrometer may be replaced with alternative AtmosphericPressure Ion Sources, Including but not limited to Atmospheric PressureChemical Ionization (APCI), Photoionization (PI), combination ES andAPCI, Desorption Electrospray Ionization (DESI) or Direct Analysis inReal Time (DART). The Electrospray ion or API source interfaced to MassSpectrometer may be replaced with alternative detectors including butnot limited to conductivity, light adsorbing and condensation nucleiparticle counting detectors. The Electrospray probe may be operated atclose to ground potential or at higher voltage. The LC or IC columnpacking material may comprise reverse phase, normal phase, ion exchangemedia or affinity chemistries. The semipermeable membranes may beconfigured with the same or different materials in the same apparatus.The second solution composition flowing through each Membrane assemblyin the same apparatus may be comprised of different compositions witheach second solution composition changing independently during a run. Inall embodiments of the invention, the sample solution flow compositioncan be changed using gradients or step functions independent of butsynchronized with other variable value changes in the same apparatus.

Embodiments of the invention can be configured to provide differentmeans of selectively capturing, releasing and/or separating samplecomponent species in the sample solution flow path. Selectivelycapturing sample components in traditional LC, IEC and Electrocapturetechniques provides a means for desalting, preconcentrating or reactingsample components prior to separation or fraction collection. Forexample, reactions with trapped or captured analyte components can beconducted by adding reagent species to effect deuterium exchange or atryptic digest of a protein. Using the invention, sample components insolution can be captured, released and/or separated and analyzed anddetected using multiple function apparatus which provides additionalcontrol of processes and new analytical methods compared to traditionalLC, IEC, CE or Electrocapture techniques. Sample components separatedusing the invention may be detected and analyzed on-line and/or fractioncollected for off-line analysis. The invention allows capture oradsorption of sample components in the solution flow using one or moreof the following methods:

-   -   1. Binding through ion-ion interaction between a solid packing        phase and the sample components in solution when using an IEC        packing media.    -   2. Adsorption between a solid packing phase and the sample        components in solution similar when using reverse phase and        normal phase chromatography packing media.    -   3. Electrocapture in open flow channels between membrane        assemblies and    -   4. Adsorption on membrane surfaces due to electrostatic forces        and/or membrane surface coatings.

The invention allows the release of adsorbed or electrocaptured samplecomponents in the sample solution flow channel using one or more of thefollowing methods:

-   -   1. Displacement of ion-ion interaction sample components by a        displacing anion or cation passing through the IEC packed column        as ion current in the sample solution flow path. The ion current        intensity, direction and composition can be changed by changing        adjacent second solution composition or relative voltages        applied to electrodes and/or by changing the sample solution        composition.    -   2. Changing pH in a packed IEC, RP or NP column by increasing or        decreasing the proton ion current (H⁺) passing through the        column in the sample solution flow path. Ion current is        increased or decreased by changing second solution compositions,        relative voltages and/or sample solution composition.    -   3. Changing organic solvent concentration in the packed or open        columns configured in the sample solution flow path by changing        the second solution solvent composition and/or the sample        solution composition.    -   4. Changing the ion current polarity and/or intensity passing        through an open channel section between membrane assemblies by        changing second solution compositions, relative voltages and/or        sample solution composition or flow rate.    -   5. Changing the ion current direction, intensity or composition        passing through a membrane by changing the membrane assembly        second solution composition or relative electrode voltages        between adjacent membrane probe assemblies.

The invention allows the separation of sample components in the samplesolution channel by:

-   -   1. Selectively adsorbing and releasing sample components in pack        columns using one or more methods listed above. Changing ion        current or ion composition through the packed columns by ramping        or stepping adjacent second solution compositions and/or        relative voltages.    -   2. Selectively Electrocapturing and releasing sample components        in open sample solution flow paths using one or more methods        listed above.    -   3. Running capillary electrophoresis in the sample solution flow        path by applying the appropriate voltages to one or more        semipermembrane assembly second solution electrodes in the        sample solution flow path or by ramping or stepping changing        second solution composition.    -   4. Running Capillary Electrochromatography in open or packed        channel sections between one or more membrane probe assemblies.

Electrospray or API source operation can be effectively decoupled fromthe upstream sample component, capture, release and separation functionsand local section ion currents by configuring the ES inlet probe with aseparate semipermeable Membrane assembly. The separate ES Membraneassembly second solution composition and second solution electrodepotential can be set to effectively isolate the ES operation fromupstream Membrane assembly generated ion currents in the sample solutionflow path. Positive or negative ion polarity can be Electrosprayed withthis invention without modifying upstream capture, release or separationconditions. This is achieved by switching the Electrospray ion sourcecounter electrode voltage polarity while applying a voltage near groundpotential to the Electrospray membrane probe assembly second solutionelectrode.

Embodiments of the invention comprising one or more membrane assemblysections, packed LC columns and/or open channel sections of the samplesolution flow path may be configured in a single integrated assemblyincluding an Electrospray inlet probe or alternatively may be configuredas discrete subassemblies. Integrated and discrete assemblies can bescaled up or down in size and sample solution flow rates to accommodatespecific analytical applications. Membrane assemblies may comprise tubeshaped or flat semipermeable membrane geometries. In alternativeembodiments of the invention, packed LC columns may be integrated intothe membrane assemblies to reduce dead volume and improve separationefficiency. In such integrated membrane and LC column embodiments, fastion current gradients are possible with the LC packing material indirect contact with semipermeable membrane. Second solution flow may beoperated in higher pressures in one or more Membrane assembly using thesample solution pressure to reference the second solution downstreampressure. This configuration minimizes or eliminates any pressuregradient from forming across a semipermeable membrane at the entranceend of packed LC or IEC column. Minimizing or eliminating the pressuregradient across a semipermeable membrane reduces the risk of membranefailure and optimizes the effectiveness of the membrane selectivity. Inembodiments of the invention, the input sample solution flow compositionmay be run isocratic or as a gradient during an LC, IEC, CE CEC orElectrocapture separation. Ion current gradients run throughsemipermeable membranes can be synchronized with a sample solutioncomposition gradient to improve LC separation efficiency.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of a packed LC column integrated into anElectrospray inlet probe positioned downstream of a single semipermeableMembrane assembly.

FIG. 2 is a diagram of one example of ion and electrical current througha semipermeable Membrane assembly interfaced to an Electrospray inletprobe.

FIG. 3 is a diagram of the ion and electrical flow through asemipermeable Membrane Electrospray probe configured with the embodimentshown in FIG. 1.

FIG. 4 is a side view cross section diagram of a Membrane assemblycomprising a tube shaped semipermeable membrane.

FIG. 5 is a front view cross section diagram of the Membrane assemblyshown in FIG. 4.

FIG. 6 is a three dimensional angled side view cross section of theMembrane assembly shown in FIG. 4.

FIG. 7 is a diagram of a packed LC column positioned between an ES inletprobe and a semipermeable Membrane assembly in the sample solution flowpath.

FIG. 8 is a diagram of two semipermeable membrane assemblies positionedupstream and downstream of a packed or open column connected on-line toan Electrospray ion source.

FIG. 9 is a diagram of ion and electrical current passing through thedual semipermeable membrane assembly with sample separation columnembodiment shown in FIG. 8.

FIG. 10 is a diagram of two semipermeable membrane assemblies configuredin series positioned downstream from a packed or open column andupstream from an Electrospray probe in an LC-ES-MS apparatus.

FIG. 11 is a diagram of three semipermeable Membrane assembliesinterfaced to an Electrospray inlet probe and configured with a sampleseparation column positioned between Membrane assemblies two and three.

FIG. 12 is a diagram of the ion current passing through upstreamMembrane assemblies two and three and the separation column in theembodiment shown in FIG. 11.

FIG. 13 is a diagram of two semipermeable membrane assemblies configuredwith a separation column, an Electrospray ion source and a lightabsorption detector.

FIG. 14 is a diagram of a two semipermeable membrane assembly with anintegrated packed separation column interfaced to an Electrospray inletprobe.

FIG. 15 is a diagram of a three semipermeable membrane assemblycomprising two open separation columns interfaced to an Electrosprayinlet probe.

FIG. 16 is a diagram of a three semipermeable membrane assemblycomprising one packed and one open separation column interfaced to anElectrospray inlet probe.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises one or more semipermeable Membraneassemblies configured along a sample solution flow path that includespacked or open channel sections. Embodiments of the invention compriseinterfacing with Electrospray inlet probes or other API source typesinterfaced to mass spectrometers. Selected ion species pass through oneor more semipermeable membranes into or from one or more second solutionflow channels and into or from a sample solution flow. Such ion speciesform an ion current along the sample solution flow path that may includea packed or open channel section or an Electrospray probe or an inletprobe to an alternative API source type. Some embodiments of theinvention comprise at least one set of adjacent semipermeable Membraneassemblies connected by a packed or opened channel sample solution flowpath. During operation, a voltage difference is maintained between theelectrodes positioned in the second solution flow channels of eachadjacent Membrane assembly. The voltage differential between Membraneassemblies drives the generation of cations or anions ions at the secondsolution electrode in the first Membrane assembly and the passing ofsuch ions through the first semipermeable membrane into the samplesolution flow path. The electric field continues to drive the cations oranions along the sample solution flow path through a packed or openchannel section. The same electric field then directs the cation oranion current from the sample solution flow path through the secondMembrane assembly semipermeable membrane to the second solutionelectrode. The cation or anion composition and current passing throughthe sample solution packed or open channel can be controlled to effectselective sample or analyte species capture, release and/or separationon line with Electrospray ionization and Mass Spectrometric analysis.Alternatively other detectors and/or fraction collectors including, butnot limited to, conductivity or light absorption detectors may beinterfaced to the semipermeable Membrane assembly apparatus to detect orcollect eluting sample species. Different embodiments of multiplesolution flow channel semipermeable Membrane apparatus and operatingmethods are described in U.S. patent application Ser. No. 11/132,953incorporated herein by reference. Although specific configurations ofsemipermeable Membrane assemblies are described in embodiments of thepresent invention, alternative semipermeable Membrane assembliesdescribed in U.S. patent application Ser. No. 11/132,953 may beconfigured as alternative embodiments of the invention.

One embodiment of the invention comprising a single Membrane assemblyand a packed separation column interfaced to an Electrospray ionizationsource is diagrammed in FIG. 1. Referring to FIG. 1, in Membraneassembly 1, sample solution flow channel 3 and second solution flowchannel 4 are separated by semipermeable membrane 2. In the embodimentshown, no electrically connected conductive surfaces are configured inthe sample solution flow path. This prevents reduction and oxidation(redox) reactions from occurring in the sample solution flow path 3during Electrospray ionization operation. Isocratic or gradient samplesolution flow is delivered by fluid delivery system or pump 16 intosample solution flow channel 3 through sample injector valve 10, Samplesolution 30 passes through flow channel 3 in contact with semipermeablemembrane 2 and flows through packed or monolithic column 9 comprisingRP, NP or IEC packing material 7, frit 11 and Electrospray tip 8. Asecond solution flow 31 is delivered from gradient fluid delivery system12, passes through second solution flow channel 4 in contact withsemipermeable membrane 2 and electrode 5 and flows into waste bottle 13.Packed separation column 9 integrated with Electrospray tip 8 isconfigured in Electrospray inlet probe 18 comprising pneumaticnebulization annulus 19 with nebulization gas inlet 20 and exit 21.Sample solution flow passing through packed or monolithic column 9 mayproduce back pressure in sample solution flow path 3. Back pressureregulator 27 references the pressure at the exit of flow channel 3 andregulates the pressure in second solution flow channel 4 to minimize oreliminate any pressure gradient across semipermeable membrane 2 duringoperation. Minimizing or eliminating any pressure gradient acrosssemipermeable membrane 2 avoids rupture of the membrane and/or reductionof its selectivity function.

Electrospray inlet probe 18 is configured in Electrospray ion source 14with endplate electrode 15, capillary orifice 17 into vacuum, capillaryentrance electrode 23 and heated counter current drying gas 22.Electrodes 5, 15, and 23 are connected to dual polarity voltage supplies24, 25, and 26 respectively. A portion of the ions generated inElectrospray source 14 are transferred into vacuum through capillaryorifice 17 where they are mass to charge analyzed.

As described in U.S. patent application Ser. No. 11/132,953, a voltageis applied between Membrane assembly electrode 5 and counter electrodes15 and 23 to form an electric field at Electrospray tip 8. For positiveion polarity Electrospray ionization, Membrane assembly electrode 5 canbe operated at or near ground potential with the endplate electrode 15and capillary entrance electrode 23 operated a negative kilovoltpotentials. Positive polarity ions entering dielectric capillary 28orifice 17 at negative kilovolt potentials can be transferred intovacuum and exit at ground or hundreds of volts above ground. Negativepolarity ions can be generated in ES ion source 14 by reversing thepolarity of each electrode. The method of changing ion potential energyin dielectric capillaries allowing Electrospray probes to be run at ornear ground potential in either ion polarity is described in U.S. Pat.No. 4,542,293 incorporated herein by reference. Alternatively, Membraneassembly electrode 5 can be operated at kilivolt potentials andcapillary entrance electrode 23 at or near ground potential providedsecond solution fluid delivery system 12 and reservoir 13 areelectrically isolated. Nozzle orifices and heated metal capillaryorifices into vacuum configured in Electrospray or other types of APIsources are typically operated at or near ground potential. Theinvention can be configured with grounded orifice into vacuum ordielectric capillary 28 whose entrance electrode 23 can be operated at avoltage range from ground to +/− kilovolt potentials. The dielectriccapillary offers performance advantages when configured with theinvention as the relative voltages between electrodes 5, 15 and 23 canremain constant or independently optimized even when stepping orscanning the voltage applied to Membrane assembly electrode 5.Consequently, the Electrospray ion source performance remains optimizedduring upstream sample species trapping, release and separationsoperation using the invention described herein.

Trapping, release and separation of sample species using the embodimentof the invention shown in FIG. 1 is achieved by ramping or stepping theElectrospray ion current, by modifying the sample solution chemistry ora combination of both. FIG. 2 is a diagram of the basic operation of theMembrane assembly 40 interfaced to Electrospray probe with pneumaticnebulization assist 41 during positive ion polarity Electrospray. Noelectrically connected conductive surfaces are configured in samplesolution flow path or Electrospray inlet probe 41. This prevents redoxreactions from occurring on conductive surfaces along the samplesolution flow path during Electrospray operation. A negative voltage isapplied to endplate or counter electrode 43 and ground or a positivevoltage is applied to Membrane assembly electrode 44 during Electrosprayoperation. The voltage difference applied between electrodes 43 and 44will range from kilovolt potentials to hundreds of volts depending onthe distance between Electrospray inlet probe tip 42 and endplateelectrode 43. The voltage difference between electrodes 44 and 43 formsan electric field at Electrospray probe tip 42 that is maintained belowthe onset of corona discharge or gas phase breakdown. The onset ofcorona discharge sets an upper bound for the relative ES voltagesapplied and constrains the ability to change total Electrospray currentby adjusting Electrospray electrode voltages. The electric fieldmaintained at Electrospray probe tip 42 is conducted through samplesolution flow channel 45, semipermeable membrane 48 and second solutionflow channel 47 to electrode 44. The total Electrospray current is afunction of the electric field at ES probe tip 42 and the totalresistance, or inversely conductivity, through the path from ES probetip 42 to Membrane assembly electrode 44. The conductivity of the liquidconductance path is changed by modifying the composition of secondsolution 50 flowing through second solution flow channel 47 and/or thecomposition of sample solution 54 flowing through channel 45. Thecomposition of second solution 50 can be changed using gradients or stepfunctions delivered from fluid delivery system or dual syringe pump 51.Syringe 52 delivers second solution 50A and Syringe 53 delivers secondsolution 50B. Each syringe delivery rate can be set or rampedindependently with manual or software control to generate any ratio ofsolutions 50A and B while retaining a constant total second solutionflow through second solution flow channel 47. In the example shown inFIG. 2, second solution 50A comprises 100% water and solution 50Bcomprises water with 10% acetic acid. As second solution 50 compositionis ramped from 100% water (only second solution 50A is delivered) toincreasing concentrations of acetic acid in water (mixture of secondsolutions 50A and B), the total Electrospray current can be scanned fromapproximately 15 nanoamps to over one microamp.

Due to the applied electric field, protons are formed throughelectrolytic processes occurring at the surface of Membrane assemblyelectrode 44. The protons formed pass through membrane 48, driven by theapplied electric field into sample solution flow channel 45. The surfaceof Membrane assembly electrode 44 may comprise, platinum, gold, carbon,stainless steel or other conductive material. Materials such as platinumor carbon are preferred to avoid electrolytic erosion of the electrodesurface during operation and to avoid generating undesired cations oranions from the electrode material that may migrate throughsemipermeable membrane 48. For the positive ion polarity examplediagrammed in FIG. 2, the total Electrospray current essentially equalsthe total proton ion current passing through cation exchange membrane48. Cation exchange membranes do not entirely block the transfer of someanions moving from sample solution 54 into second solution 47, driven bythe electric field, but the contribution to the total current passingthrough electrode 44 is quite small. In the example shown, cationexchange membrane 48 comprises sulfonated fluoroethylene material(perfluorosulfonic acid polytetrafluoroethylene (PTFE) copolymer) oneformulation of which is Nafion® ® Dupont). Other membrane materials canbe configured in Membrane assemblies including but not limited tocellulose esters, polysulfone dialysis tubing with different molecularweight cutoffs, cation or anion exchange semipermeable membranesavailable from Dionex Corporation and cation exchange membranes from RAIResearch Corporation (Raipore R4010 and R1010). As diagrammed in FIG. 2,electrolysis occurring at the surface of Membrane assembly electrode 44produces protons that are transferred through cation exchange membrane48 driven by the electric field into sample solution flow channel 45.The protons move with sample solution flow channel 45, driven by theflow of sample solution 54 and the Electrospray electric field, and exitthrough Electrospray probe tip 42. The protons formed at Membraneassembly electrode 44 provide essentially the total Electrospray chargeddroplet current formed in Electrospray 55. Consequently, as the totalElectrospray current increases, the pH of sample solution 54 decreasesin sample solution flow channel 45 between semipermeable membrane 48 andElectrospray probe tip 42.

PH scans in sample solution 54 can be conducted from lower to higher pHor higher to lower pH by running a gradient or step function of acidconcentration in second solution 50. PH is defined as the log of protonor H+ concentration or molarity in solution, so a ten times increase inElectrospray total ion current corresponds to a one unit drop on the pHscale. Running Electrospray with pneumatic nebulization assist, a rangeof total Electrospray current covering over three orders of magnitudecan be achieved using the embodiment of the invention as shown in FIG.2. Sufficient membrane area in contact with sample solution 54 andsecond solution 50 is needed to support higher current operation.Scanning Electrospray total ion current over three orders of magnitude,the pH in the sample solution is scanned over a 3 pH units range. Forexample, if sample solution 54 is 100% aqueous, a pH scan in the samplesolution can range from just below pH 7 to pH 4. When negative ionpolarity Electrospray is run using a cation exchange membrane 48,protons are removed from sample solution 54 as it flows through samplesolution flow channel 45. As the negative ion polarity totalElectrospray current is increased, the pH in sample solution 54increases. In the case of a 100% aqueous sample solution 54 a pH scanranging from approximately 7 to above 9 could be conducted by increasingthe acid concentration or conductivity of second solution 50. If it isdesirable to scan through a different region of the pH scale, the samplesolution can be buffered to a desired pH with an appropriate bufferconcentration added to sample solution 54. Increasing the Electrospraycurrent while running the buffered sample solution, by increasing acidconcentration in second solution 50, changes the pH from the initialbuffered pH value.

Alternately, concentrations of bases or salts added instead of acids tosecond solution 50 can be changed by running concentration gradientsflowing through second solution flow channel 45. By selecting theappropriate cation or anion exchange membrane and appropriate solvents,specific cations such as sodium or potassium can be transferred throughsemipermeable membrane 48 instead of protons. For many Electrosprayapplications, sodium or potassium may not be the preferred chargecarrier species but, as will be described for different embodiments ofthe invention, such cations may be a preferred species to displace boundsample components when IEC packing materials are used. Controllingsample solution pH can also be used in certain applications to initiallypromote capture or binding of sample species on an RP or IEC packingmaterial and subsequently releasing the bound sample species by changingthe solution pH. Using the embodiment shown in FIG. 1, packing material7 can be selected to binding sample species at an initial pH establishedby the Electrospray current and release the bound sample species at adifferent pH established using a different Electrospray current. Thismethod can be used for cleanup, desalting or separation of samplecomponents. Bound sample can also be subjected to reactions by changingthe sample solution composition and maintaining constant totalElectrospray current. Alternatively, the conductivity and organicsolvent concentration can be stepped or ramped in second solution 50.With the selection of the appropriate semipermeable membrane, organicsolvent will pass through the membrane driven by concentration gradientsacross the membrane to improve the efficiency or release of samplespecies bound to packing material 7, complimenting or enhancing a pHgradient. The composition of sample solution 54 and second solution 50can be changed independently to most efficiently accommodate onlinesample cleanup and/or separation of sample species in the embodiment ofthe invention shown in FIG. 1.

The ion and electrical current pathways occurring in the embodiment ofthe invention shown in FIG. 1 are diagrammed in FIG. 3 for the case ofpositive ion Electrospray operation from an aqueous solution with protontransfer through semipermeable membrane 2. The same numbers are used toidentify the same elements in FIGS. 1 and 3. In the embodiment of theinvention diagrammed in FIG. 3, no conductive surfaces are configured insample solution flow path 3 preventing redox reactions from occurring onsurfaces in sample solution flow path 3. Proton charge transfer throughcation exchange membrane 2 is diagrammed in FIG. 3 to illustrate protonion current passing from second solution 58 into the sample solution 57.The total Electrospray current I_(H1) comprising protons and indicatedas 61 is equal but opposite in to the electron current I_(e1) indicatedas 60 transferred from Membrane assembly electrode 5 into power supply24. I_(e1) equals the sum of I_(e2), I_(e3) and I_(I4) indicated as 62,63 and 64 respectively. The electron current I_(e2), and I_(e3) and ioncurrent I_(I4) is equal to the fraction of the total charge I_(H1)Electrosprayed from tip 8 that impinges on endplate electrode 15(I_(e2)), capillary entrance electrode 23 (I_(e3)) or passes into vacuumthrough capillary orifice 17 (I_(I4)) respectively. I_(H1) impinges onelectrodes 15 and 23 or passes into vacuum as gas phase ions, chargeddroplets or charged solid aerosols produced in the Electrospray process.Monitoring electron current on electrode elements 5, 15 and 23 duringoperation provides a means of monitoring and controlling theElectrospray ion current and the pH or cation or anion concentration insample solution flow channel 3 during Electrospray operation in theembodiment shown in FIG. 3 and other embodiments of the inventiondescribed below.

Second solution 58 flowing through second solution flow channel 4provides a means for running Electrospray current and pH gradients andprevents the depletion of charge which can occur in static reservoirs inelectrolytic cells configured and operated with charge removal throughsemipermeable membranes. It has been found that the flow rate of secondsolution 58 through second solution flow channel 4 needed to maintain asteady Electrospray current is less than the flow rate of samplesolution 57 through sample solution flow channel 3. Depending on thesample solution flow rate and the total Electrospray current run, aminimum second solution flow rate is required to maintain steady andstable conditions. Running the second solution flow rate higher than theminimum required level did not improve performance or change the totalES current. Consequently, running the second solution flow rate justabove the minimum required value for a given application greatly reducessecond solution consumption during operation. However, operating withhigher second solution flow rates through channel 4 allows the runningof faster ion current or pH gradients. Second solution flow channel 4can be configured according to the invention with low dead volume toallow the running of precise and/or rapid gradients with minimum secondsolution consumption.

Membrane assembly 1 and 40 diagrammed in FIGS. 1 through 3 comprises aflat sheet semipermeable membrane geometry with low dead volume solutionflow channels configured on opposite sides of the membrane.Semipermeable membranes 2 or 48 clamped between opposite electricallyinsulating body elements 65 and 66 can serve as a liquid seal oradditional elements 6 can be configured in Membrane assembly 1 to serveas seals to prevent leakage from sample solution 57 or second solution58, particularly for higher pressure applications. Higher pressures canoccur in sample solution flow channel 3 due to sample solution flowthrough packed or monolithic column 9. As was described above, thepressure gradient across semipermeable membrane 2 can be minimized byincluding back pressure regulator 27 downstream in second solution flow4 that references the pressure in sample solution flow channel 3 or byother means known in the art.

An alternative embodiment of the Membrane assembly is diagrammed inFIGS. 4, 5 and 6. FIG. 4 is a cross second side view of Membraneassembly 80 interfaced with Electrospray ion source 14 and mass tocharge analyzer and detector 83. FIG. 5 is a cross section front view ofMembrane assembly 80 and FIG. 6 is an angle three dimensional crosssection view of the center region of Membrane assembly 80. Commonelements in FIGS. 4, 5 and 6 share common numbers and element common into those in FIG. 1 have the same number. Referring to FIGS. 4, 5 and 6,Membrane assembly 80 comprises tube shaped semipermeable membrane 70 andsample solution flow tube sections 85 and 86. As shown in FIG. 6,semipermeable membrane 70 is configured in a sleeve tubes 90 and 91which form a seal with Membrane assembly body 87 using compressionferrules 92 and 93 respectively. Alternatively semipermeable membranemay be sealed to Membrane assembly body 87 with ferrules compresseddirectly on semipermeable membrane 70 or semipermeable membrane 70 canbe bonded and sealed to Membrane assembly body 87 using an appropriatebonding agent. Gap 84 is configured between sample solution flow tubes85 and 86 to allow sample solution 88 to contact semipermeable membrane70 as it flows through Membrane assembly 80. Second solution 89 flowsthrough second solution flow channel 72. Gradient fluid delivery pump 12provides second solution flow, through Membrane assembly 80 and backpressure regulator 27, into reservoir 13. Gradient fluid delivery pump12 may be configured as, but is not limited to, a dual syringe pump,dual piston pump, peristaltic pump or a diaphragm pump. Second solutionflow channel 72 passes around tube shaped semipermeable membrane 70 incontact with second solution electrode 82. Second solution electrode 82is connected to dual polarity power supply 24. Sample solution 88 flowis delivered from isocratic or gradient pump or pressurized reservoir 16through sample injector valve 10, tube 86, gap 84, tube 85 and exits atElectrospray tip 75. Electrospray probe 73 with pneumatic nebulizationassist comprises tube 74 with Electrospray tip 75 and nebulizer gasinlet flow 76 and outlet flow 77. Charged droplets formed inElectrospray plume 78 are directed by the Electrospray electric field tomove toward the entrance of capillary orifice 17 against heatedcountercurrent drying gas 22. A portion of the ions formed are sweptinto vacuum 81 through capillary orifice 17 and are mass to chargedanalyzed by mass to charge analyzer and detector 83. Vacuum system 81may comprise one or multiple vacuum stages as is known in the art.

Referencing the embodiment of the invention diagrammed in FIGS. 4, 5 and6, the surface area of semipermeable membrane 70 in contact with samplesolution 88 can be increased or decreased by moving sample solution flowtubes 85 and 86 further apart or closer together respectively.Increasing the semipermeable membrane surface area in contact withsample solution 88 will allow increased ion current capacity required insome applications. The operation of the Membrane assembly shown in FIGS.4, 5 and 6 is analogous to the flat sheet Membrane assemblies 1 and 40diagrammed in FIGS. 1, 2 and 3. The voltage difference applied betweensecond solution electrode 82 and end plate electrode 15 and capillaryentrance electrode 23 establishes the electric field at Electrospray tip75. The electric field extends from Electrospray tip 75 through samplesolution 88, semipermeable membrane 70 and second solution 89 to secondsolution electrode 82. The total Electrospray current can be controlledby changing the composition of second solution 89, sample solution 88 orto a limited extent by changing the applied Electrospray voltage butremaining below the onset of corona discharge at Electrospray tip 75. PHscans can be run in sample solution 88 can by ramping or stepping theconductivity of second solution 89. As described above, pH or cation oranion current scans can be run to effect binding, release and/orseparation of sample species in RP, NP or IEC packed media inElectrospray needle 74 or in an LC or IEC column configured in samplesolution flow path 71 between Membrane assembly 80 and Electrosprayprobe 73 as diagrammed in FIG. 7.

The sample solution flow path in Membrane assembly 80 and Electrosprayprobe 73 are configured with no electrically connected conductivesurfaces in the sample solution flow path. When injector valve 10 isoperated at ground potential during Electrospray operation, the voltageapplied to second solution electrode 82 can be adjusted to null anyelectric field from extending through sample solution flow path 71upstream of Membrane assembly 80. This prevents any redox reactions fromoccurring on upstream grounded conductive surfaces in injector valve 10or fluid pump 16 that are in contact with the sample solution. Theelimination of redox reactions occurring on conductive surfaces in thesample solution flow path minimizes or eliminates any redox reactionbased changes to sample species and avoids the plating of anion orcation species on upstream surfaces. When the Electrospray ion polarityis switched such plated cation or anion species will reenter the samplesolution flow causing unwanted contamination peaks in the mass spectraand potentially compromising Electrospray performance.

The tube or flat sheet semipermeable membrane configurations areanalogous in function but the geometry of one may have an advantage overthe other in specific applications. For example, flat sheetsemipermeable membranes may be configured with less sample solutionchannel volume compared with tube shaped semipermeable membraneassemblies. Flat sheet semipermeable membranes provide simpler compactlayered flow path geometries as shown in the embodiment of the inventiondiagrammed in FIG. 14. Tube shaped semipermeable membrane assemblies mayprovide a geometry advantage when minimum linear contact distancebetween the sample solution and semipermeable membrane along thesolution flow path is desired to effect the highest separationefficiency of sample species. The round tube and flat sheetsemipermeable Membrane assembly embodiments shown in FIGS. 1 through 6can accommodate thicker or thinner semipermeable membranes. Membranes ofsimilar or different material can be stacked or sleeved to improvecation or anion transfer selectivity.

An alternate embodiment of the invention is diagrammed in FIG. 7.Similar elements diagrammed in FIGS. 1 and 7 retain the same numbers andfunction as describe previously. In the embodiment shown in FIG. 7,packed LC or IEC column 100 is configured in sample solution flow path 3between Membrane assembly 1 and Electrospray probe 101. Packed column100 is analogous in function to packed column 9 shown in FIG. 1 butallows additional flexibility in accommodating different column sizes,packing materials, column materials, commercial availability and samplesolution flow rates. A second heated gas layer is configured inElectrospray probe assembly 101 to facilitate drying of Electrosprayedliquid droplets. Nebulization gas enters Electrospray probe at gasentrance 110 and exits at 111 through an annulus surroundingElectrospray tip 114. Heated gas flow enters the second gas layer at 112passes through gas heater 109 and exits at 113. Controller 107 connectedto power supplies 24, 25 and 26 and fluid delivery systems 12 and 16 canbe programmed to provide independent but synchronized control ofvoltages and sample solution and second solution gradients to optimizesample component cleanup and/or separation and Electrospray performance.

Two Membrane assemblies are configured in series in an alternativeembodiment of the invention diagrammed in FIGS. 8 and 9. Common elementsdescribed from alternative embodiments retain the same numbers.Referring to FIGS. 8 and 9, sample solution flow channels 125 and 4 ofMembrane assemblies 121 and 118 respectively are configured in serieswith packed column or open channel 120 positioned in sample solutionflow channel 131 connecting sample solution flow channels 125 and 3.Membrane assemblies 121 and 118 can be configured comprising flat sheetsemipermeable membranes as diagrammed in FIGS. 1, 2 and 3 or comprisingtube shaped semipermeable membranes as diagrammed in FIGS. 4, 5 and 6.Membrane assembly 121 comprises flat sheet or tubular semipermeablemembrane 130, sample solution flow channel 125, second solution flowchannel 126, second solution electrode 127, dual polarity power supply122, second solution gradient fluid delivery pump 123, second solutionoutlet reservoir 133 electrically insulating and chemically inert body134 and second solution back pressure regulator 124. Similarly Membraneassembly 118 comprises flat sheet or tubular semipermeable membrane 2,sample solution flow channel 3, second solution flow channel 4, secondsolution electrode 5, second solution outlet reservoir 13, dual polaritypower supply 24 and second solution gradient fluid delivery system 12.Membrane assembly 118 sample solution flow channel 3 outlet isinterfaced to Electrospray probe 101. Column 120 may be packed with RP,NP or IEC media or may be configured as an open channel column to allowElectrocapture, capture, release or separation functions or capillaryelectrophoresis separation methods to be run. In the embodiment shown inFIGS. 8 and 9, no electrically conductive surfaces are configured in thesample solution flow path to prevent redox reactions from occurring onconductive surfaces along the sample solution flow path. Elements ofsample injection valve 10 and gradient pump 16 that contact the samplesolution comprise electrically insulating materials or are electricallyfloated during operation. All ion current passing through the samplesolution flow path and the total Electrospray current must betransferred through semipermeable membranes 134 and 2 during operation.

Adding Membrane assemblies along the sample solution flow path increasesthe analytical flexibility and capability of LC, IEC, CE andElectrocapture apparatus. Sample species injected into the samplesolution flow through sample injection valve 10 enter Membrane assembly121 as diagrammed in FIG. 9. Relative voltage amplitudes and polaritiesapplied to Membrane assembly 118 second solution electrode 5 andendplate electrode 15 and capillary entrance electrode 23 and thecomposition of second solution 58 can be adjusted as independentvariables to optimize Electrospray and ES MS performance during ananalysis as was described above. Upstream sample capture and releasesteps conducted in column 120, and used for sample concentration, samplecleanup, conducting reactions with sample species and/or separationfunctions, can be controlled independently of downstream Electrosprayoperation. Variables used to control functions occurring between theinlet of Membrane assembly 121 and the outlet of Membrane assembly 118include but are not limited to:

-   1. The relative voltage polarity and amplitude applied between    second solution electrodes 127 and 5,-   2. Semipermeable membrane 2 composition (cation or anion exchange    membrane),-   3. Semipermeable membrane 130 composition (cation or anion exchange    membrane),-   4. Second solution 58 composition, (isocratic, step function or    gradient),-   5. Second solution 129 composition, (isocratic, step function or    gradient),-   6. Column 120 configuration, (packed or open channel),-   7. Column 120 packing media, (including but not limited to size    exclusion, reverse phase, normal phase or ion exchange    chromatography media),-   8. Sample solution flow rate, and-   9. Sample solution composition, (isocratic, step function or    gradient).

The above listed variables can be manually controlled or synchronouslycontrolled through software using controller 135 to conduct one or moreof the following analytical functions when a mixture of samplecomponents is injected into sample solution flow 128 through sampleinjection valve 10:

-   1. Selective capture and release of sample species on semipermeable    membrane 130,-   2. Selective capture and release of sample species on the stationary    phase of packed column 120 through RP, NP, size exclusion or IEC    binding of sample species to the packed media,-   3. Selective capture and release of sample species in open channel    120 using Electrocapture,-   4. Separation of sample species in open channel 120 using Capillary    Electrophoresis,-   5. Selective capture and release of sample species on semipermeable    membrane 2,-   6. Preconcentration, desalting or cleanup of captured samples prior    to release of sample species,-   7. Reaction of captured sample species with chemical species    introduced through injection valve, 10 or through changing sample    solution composition delivered by pump 16 through gradients or step    functions, and/or-   8. Separation of species through controlled release of samples    trapped on semipermeable membranes 134 or 2 or in column 120.

FIG. 9 illustrates an example of capture and selective release of samplespecies in column 120. A mixture of sample components C₁, C₂ and C₃ isinjected into sample solution flow 128 through sample injector valve 10.Semipermeable membranes 2 and 130 comprise proton membranes.

Little or no ion current initially passes through column 120 by settingthe relative voltages applied to electrodes 127 and 5 to a value thatzeros the electron current I_(e2). The sample solution composition isselected to promote binding of sample species on column 120 ion exchangepacking material. Bound sample species are then selectively released byincreasing the proton current passing between Membrane assemblies 118and 121. Protons passing through IEC column 120 change the pH to effectsample species release or the protons serve as a cation displacer ofbound samples. A pH ramp can be run to effect separation of componentspecies with subsequent on line Electrospray ionization and MS analysis.The proton current amplitude and direction through column 120 can bechanged by increasing the relative voltage amplitude and polarityapplied to electrodes 5 and 127. The proton current amplitude can alsobe ramped or stepped by changing the composition of one or both secondsolutions 58 and 129.

As a second example, column 120 can be configured as an open channel andinjected sample components can be Electrocaptured by maintaining anappropriate voltage difference and polarity between electrodes 5 and 127and controlling the sample solution flow rate. Selective release orseparation of Electrocaptured sample components can be achieved byapplying one or more of the following methods:

-   1. Ramping or stepping the composition of one or both second    solutions 58 and/or 129 to change the ion current passing through    open channel 120.-   2. Changing the relative voltage amplitude or polarity applied to    electrodes 5 and 127.-   3. Change the sample solution flow rate and/or composition.

As a third example, sample components that have a net charge in solutioncan be trapped at the surface of semipermeable membrane 130 in contactwith the sample solution by the applying the appropriate electric fieldamplitude and polarity. Trapped samples can be concentrated, desaltedand subjected to reactions with components added to the sample solution.The relative voltages applied between electrodes 5 and 127 and thecomposition of second solutions 58 and 129 establish the polarity andamplitude of the electric field at the surface of semipermeable membrane130. The polarity of this electric field can be reversed as a gradientor step function to release sample components trapped on the surface ofsemipermeable membrane 130 followed by on-line Electrospray MS analysis.

The relative voltages applied between electrodes 5, 15 and 23 can beheld constant to provide consistent Electrospray performance even whilechanging upstream relative voltages applied between electrodes 5 and127. Dielectric capillary 28 allows the absolute voltage amplitudes andpolarities applied to electrodes 5, 15 and 23 to range over thousands ofvolts while maintaining the relative voltages and Electrosprayperformance constant. This allows electrode 129 to be set at or nearground potential to avoid redox reactions from occurring on anyelectrically connected or grounded conductive surfaces along the samplesolution flow path. By maintaining electrode 127 at ground potential, noconstraint is placed on upstream component materials and electricalisolation of electrically conductive surfaces is no longer required.Standard commercially available injection valves or pumps can beconfigured in the embodiment shown in FIGS. 8 and 9. Cation or anioncurrents passing through packed column or channel 120 can be directlymonitored and controlled by measuring electron currents I_(e2) andI_(e1). As described above, the total Electrospray current can bemonitored and controlled by measuring electron currents I_(e1), I_(e3)and I_(e4). The total Electrospray current will equal I_(e1) minusI_(e2). The ion current passing through packed column or open channel120 adds no cation or anion concentration to the sample solution flowexiting Membrane assembly 118. All ion current transferred through onesemipermeable membrane that passes through packed column or open channel120 is removed through the second sample solution by passing through thesecond semipermeable membrane.

For selected applications, to avoid running a Membrane assembly athigher pressures, packed column or open channel 138 can be configuredupstream of Membrane assembly 121 as diagrammed in FIG. 10. Similarelements or assemblies described in alternative embodiments areidentified by the same number. Referring to FIG. 10, ion current passingthrough packed or open channel column 138 is generated at the surface ofsecond solution electrode 127 in Membrane assembly 121 and passesthrough semipermeable membrane 130. Grounded union or junction 139completes the ion current circuit from electrode 127. The appliedelectric field extending through column 138 and the ion currentamplitude and direction between grounded union 139 and electrode 127 iscontrolled by the voltage amplitude and polarity applied to electrode127, the composition of second solution 129 and the composition ofsample solution 128. The relative voltage amplitude and polarity appliedbetween second solution electrodes 5 and 127 can be set to minimize ioncurrent through sample solution flow channel 131. This effectivelyseparates control, composition and polarity of total Electrospray ioncurrent and the ion current passing through column 138. Theconfiguration of ground union 139, however, causes redox reactions tooccur on a conductive surface in the sample solution flow channel. Byproducts of such reactions may remain in the sample solution flow path.For example, if sodium cations are transferred though semipermeablemembrane 130, are directed through column 138 as ion current and areneutralized on conductive union 139, the neutralized sodium remains inthe sample solution flow path which may compromise Electrosprayperformance. If proton ion current is neutralized on conductive surface139, hydrogen gas may form in the sample solution flow path which maydisrupt downstream processes.

To improve analytical performance, capability and flexibility whilepreventing redox reactions from occurring in the sample solution flowpath, three Membrane assemblies can be configured in series along thesample solution flow path as shown in the alternative embodiment of theinvention diagrammed in FIGS. 11 and 12. Packed or open Channel column138 is positioned upstream in the sample solution flow path fromMembrane assemblies 121, 118 and Electrospray probe 101. Sample injectedthrough sample injection valve 10 flows though Membrane assembly 140sample solution flow channel 143, packed column or open channel 138,Membrane assembly 121 sample solution flow channel 125, connectingchannel 131, Membrane assembly 118 sample solution flow channel 3 and isElectrosprayed from Electrospray probe 101 with subsequent mass tocharge analysis and detection. Similar to two Membrane assemblyembodiments, back pressure regulator 145 referencing the pressure insample solution flow channel 143 minimizes the pressure differentialacross semipermeable membrane 141 configured in Membrane assembly 140.Dual polarity power supply 157 supplies voltage to second solutionelectrode 148 and gradient pump 144 delivers second solution flow withisocratic or varying composition to second solution flow channel 142.

The three Membrane assembly embodiment diagrammed in FIG. 11 allowsindependent control of the upstream sample component capture, releaseand separation functions and the downstream the Electrospray process.FIG. 12 shows an example operating mode where protons are generatedthrough oxidation reactions occurring at the surface of second solutionelectrode 127 and are transferred through cation exchange membrane 130into sample solution flow channel 127. Proton ion current passes throughpacked column or open channel 138 driven by the electric fieldmaintained between second solution electrodes 127 and 148 in Membraneassemblies 121 and 140 respectively. Protons then pass through cationexchange membrane 141 and are reduced on second solution electrode 148configured in Membrane assembly 140.

The relative voltage amplitude and polarity applied to second solutionelectrodes 5 and 127 can be controlled to minimize or prevent upstreamion current from passing through sample solution flow channel 131.Alternatively, the composition, amplitude and direction of ion currentpassing through sample solution flow channel 131 can be controlled byusing one or more of the following variables:

-   1. The relative voltage polarity and amplitude applied between    second solution electrodes 127 and 5,-   2. Semipermeable membrane 2 composition (cation or anion exchange    membrane),-   3. Semipermeable membrane 130 composition (cation or anion exchange    membrane),-   4. Second solution 58 composition, (isocratic, step function or    gradient),-   5. Second solution 129 composition, (isocratic, step function or    gradient)-   6. Sample solution flow rate, and-   7. Sample solution composition, (isocratic, step function or    gradient).

As described above for the two Membrane assembly embodiment shown inFIG. 8, Electrocapture sample component preconcentration, capture,release and separation methods can be run in sample solution flowchannel 131 by controlling the electric field and ion current amplitudeand direction and the sample solution flow rate in sample solution flowchannel 131. Depending on the application requirements, one ion speciesof anions or cations can be generated and directed through packed columnor open channel 138 and a different ion species of anions or cations canbe generated to pass through sample solution flow channel 131. Ioncurrent directed through pack column or open channel 138 and samplesolution flow channel 131 can be controlled to have:

-   1. The same or different amplitude,-   2. The same or different direction,-   3. The same or different cation or anion generation source,-   4. The same or different cation or anion species, or-   5. The same or different ion polarity.

Each of the above conditions can remain static or be changed during arun. Ion current composition, amplitude and direction through packedcolumn or open channel 138 can be controlled using one or more of thefollowing variables:

-   1. The relative voltage polarity and amplitude applied between    second solution electrodes 127 and 148,-   2. Semipermeable membrane 130 composition (cation or anion exchange    membrane),-   3. Semipermeable membrane 141 composition (cation or anion exchange    membrane),-   4. Second solution 129 composition, (isocratic, step function or    gradient),-   5. Second solution 155 composition, (isocratic, step function or    gradient),-   6. Column 138 configuration, (packed or open channel),-   7. Column 138 packing media, (including but not limited to size    exclusion, reverse phase, normal phase or ion exchange    chromatography media),-   8. Sample solution flow rate, and-   9. Sample solution composition, (isocratic, step function or    gradient).

The voltage amplitude and polarity applied to second solution electrode148 can be set close to ground potential to prevent redox reactions fromoccurring on upstream conductive surfaces. All other downstream andElectrospray source electrodes can be adjusted to optimize desiredsample component preconcentration, desalting, cleanup, separation orreactions with reagent species functions and Electrospray ionizationmass analysis performance.

The above listed variables can be manually controlled or synchronouslycontrolled through software using controller 135 to conduct one or moreof the following analytical functions when a mixture of samplecomponents is injected into the sample solution flow through sampleinjection valve 10:

-   1. Selective capture and release of sample species on semipermeable    membrane 148,-   2. Selective capture and release of sample species on semipermeable    membrane 130,-   3. Selective capture and release of sample species on semipermeable    membrane 2,-   4. Selective capture and release of sample species on the stationary    phase of packed column 138 through RP, NP, size exclusion or IEC    binding of sample species to the packed media,-   5. Selective capture and release of sample species in open channel    138 using Electrocapture,-   6. Selective capture and release of sample species in open channel    131 using Electrocapture,-   7. Preconcentration, desalting or cleanup of captured samples prior    to release of sample species in packed column or open channel 138 or    open channel 131,-   8. Reaction of captured sample components with reagent chemical    species introduced through injection valve 10 or through changing    sample solution composition delivered by pump 16 through gradients    or step functions in packed column or open channel 138 or open    channel 131, and/or-   9. Separation of species through controlled release of samples    trapped on semipermeable membranes 141, 130 and/or 2, in column or    open channel 138 or in open channel 131.

Electron currents 150 and 151 can be monitored to control the ioncurrent 152 through packed column or open channel 138 or ion current 158through open channel 131. All ion current passing through packed columnor open channel 138 can be removed from the sample flow solution beforethe sample flow solution passes through open channel 131 by controllingelectron currents 150 and 151 to be equal but opposite in direction andzeroing ion current 158 using methods described above. For examplesodium cations can be used to displace sample species from ion exchangemedia packed in column 138 and removed from the sample flow solutionupstream of Membrane assembly 2 as sodium ion current would reduceElectrospray performance. Simultaneously and independently, protons canbe generated in Membrane assembly 118 to provide the Electrospray ioncurrent. Specifically in this example, a sodium cation current isgenerated at the surface of electrode 148 in second solution 155 ofMembrane assembly 140 and directed through semipermeable membrane 141configured as a cation exchange membrane. Driven by the electric fieldmaintained along the sample solution flow path by voltages applied tosecond solution electrodes 148 and 127, the sodium cation current passesthrough packed column 138 displacing sample species captured on the ionexchange media packed in column 138. After exiting column 138, excesssodium cations are removed from the sample solution flow, by passingthrough semipermeable membrane 130 configured as a cation exchangemembrane, driven by the electric field, and reduced or neutralized atthe surface of second solution electrode 127. Protons are independentlygenerated at the surface of second solution electrode 5 throughoxidation reactions driven by the Electrospray electric field and aretransferred through semipermeable membrane 2 configured as a cationexchange membrane to provide the Electrospray current. Multidimensionalpreconcentration, cleaning, and separations of sample species can beperformed by synchronizing the capture, release and separation functionsconducted in packed column or open channel 138 and those conducted inopen channel 131.

All operating modes of the three Membrane assembly embodiment of theinvention can be controlled manually or synchronously through softwarecontrol through controller 135. Different detectors can be used todetect sample components exiting Membrane assembly 118 as diagrammed inFIG. 13. Separation of sample species in packed column or open channel120 has been described above. Separated sample components exit Membraneassembly 118 carried by the sample solution flow through channel 160. Inthe embodiment of the invention diagrammed in FIG. 13, the samplesolution flow is split in flow splitter 161 with a portion of samplesolution flow directed though Electrospray inlet probe 101 with theremainder directed through UV detector flow cell 162. Sample solutionpasses through UV detector flow cell 162 and is fraction collected infraction collector 163. UV detector electronics 164 and logic interface165 send the digitized UV detector signal to controller 135. The samplesolution flow path through UV detector flow cell 162 and fractioncollector 163 is electrically isolated to avoid redox reactions fromoccurring in the sample solution flow path due to any Electrosprayelectric field. UV detector 162 can be any type of detector used in LC,IEC, CE or CEC including but not limited to variable or multiplewavelength light adsorption detectors (photodiode arrays), conductivitydetectors or condensation nuclei particle counting detectors. Other massspectrometer ion sources including but not limited to AtmosphericPressure Chemical Ionization (APCI), Photoionization, InductivelyCoupled Plasma (ICP), or combination Electrospray and APCI sources canbe used instead of Electrospray ion sources interfaced to massspectrometers.

In an alternative embodiment of the invention, one or more Membraneassemblies, packed columns and/or open channels can be configured in asingle integrated assembly. Integrated assemblies can be configured tominimize size and sample solution flow channel dead volumes and flowrates. An integrated two Membrane assembly embodiment of the inventionis diagrammed in FIG. 14 wherein packing or monolithic sample separationmedia 176 is configured in the sample solution flow path 177 in contactwith semipermeable membrane 172. Dual Membrane assembly 170 comprisesdownstream semipermeable membrane 171, sample solution flow channel 173,second solution flow channel 174 and second solution electrode 187 andupstream semipermeable membrane 172, sample solution flow channel 177,second solution flow channel 175 and second solution electrode 188.Solution flow channel 173 is integrated into Electrospray probe 178whereby no conductive surfaces are configured in the sample solutionflow path to prevent redox reactions from occurring in the samplesolution flow path. Dual Membrane assembly 170 can be run in operatingmodes similar to those described for the two Membrane assemblyembodiment of the invention diagrammed in FIG. 8. Open channel 185 canbe run in Electrocapture and release mode as a compliment or in serieswith chromatography functions run using packed or monolithic media 176.Efficient and rapid displacement of selected components trapped on media176 can be achieved, to effect separation of sample components, due tothe close proximity and contact of media 176 to semipermeable membrane172. Ions passing through semipermeable membrane 172 are in contact witha large portion of the packing or monolithic separation media 176 sotightly controlled ion currents can provide a nearly uniform chemicalenvironment throughout the volume of packing or monolithic material 172.Ion currents 183 passing through sample solution flow channel 185 andthe total Electrospray current 184 can be controlled by monitoringelectrical currents 179, 180, 181 and 182 and controlling voltagesapplied to electrodes, second solution composition, sample solutioncomposition and flow rate, semipermeable membrane materials and packingor monolithic separation media composition as has been described foralternative embodiments above. Dual membrane assembly 170 can be readilyscaled up or down in size and alternatively configured with tubularinstead of flat sheet semipermeable membrane elements. Additionalsemipermeable membrane layers can be added to an integrated assembly toincrease analytical functional capability, performance and flexibility.

FIG. 15 shows a diagram of a four Membrane assembly embodiment of theinvention that can be configured with discrete components and assembliesor as one integrated assembly. Four Membrane assembly apparatus 200comprises Membrane assemblies 201, 202, 203 and 204 with second solutiongradient pumps 215, 216, 217 and 218 respectively and second solutionoutlet reservoirs 220, 221, 222 and 223 respectively. Sample solution isdelivered using gradient pump 219 and sample is injected through sampleinjection valve 224. The four Membrane assembly apparatus is interfacedto Electrospray probe 207 and mass spectrometer 208 with all functionssynchronized and controlled through controller 210. Two open channelsections 205 and 206 are sequentially positioned along the samplesolution flow path between Membrane assemblies 221 and 222 and 222 and223 respectively. The four Membrane assembly embodiment diagrammed inFIG. 15 allows multidimensional trapping, release and separationanalytical functions as described above conducted online withElectrospray mass spectrometric analysis. An extended range ofanalytical methods can be programmed and controlled in a modular fashionusing controller 210. Components can be swapped out or added to rapidlyreconfigure system as diagrammed in FIG. 16 wherein a packedchromatography column 212 replaces open channel section 206 in fourMembrane assembly apparatus 211. Back pressure regulator 213 is added tominimize any pressure gradient across the semipermeable membraneconfigured in Membrane assembly 204. Using the embodiment of theinvention diagrammed in FIG. 16, orthogonal sample preconcentration,reaction, cleaning and/or separations can be performed through thepacked RP, NP, IEC or CEC column 212 and open channel 205.

Alternative embodiments of Membrane assemblies and combinations ofMembrane assemblies, ion sources, detectors and analyzers can beconfigured, including but not limited to parallel Membrane assemblyconfigurations and using gas phase ion mobility detectors or ionmobility detectors interfaced to mass spectrometers. Although thepresent invention has been described in accordance with the embodimentsshown, one of ordinary skill in the art will recognized that there canbe variations to the embodiments, and those variations would be withinthe spirit and scope of the present invention.

It should be understood that the preferred embodiment was described toprovide the best illustration of the principles of the invention and itspractical application to thereby enable one of ordinary skill in the artto utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. All suchmodifications and variations are within the scope of the invention asdetermined by the appended claims when interpreted in accordance withthe breadth to which they are fairly legally and equitably entitled.

1.-25. (canceled)
 26. An apparatus for analyzing chemical speciescomprising: a. at least one membrane assembly each comprising asemipermeable membrane, a first flow channel having a first flow channelinlet and a first flow channel outlet, a second flow channel having asecond flow channel inlet and a second flow channel outlet, saidsemipermeable membrane separating said first and second flow channels,b. a first and a second solution flowing in said first and second saidflow channels, respectively; c. means for introducing sample speciesinto said a sample solution flowing through said first flow channel; d.a detector responsive to at least a portion of said sample speciesflowing from said first solution flow outlet of one of said membraneassemblies and; e. at least one means for adding chemical componentsinto or eliminating chemical components from said first solution flowthrough said at least one semipermeable membrane.
 27. An apparatusaccording to claim 26 wherein an electrode is positioned in said secondflow channel.
 28. An apparatus according to claim 26, further comprisinga means for applying and controlling voltages applied to said electrode.29. An apparatus according to claim 26, further comprising means forindependently changing the composition of said first and secondsolutions.
 30. An apparatus according to claim 26 wherein said detectorcomprises an ion source for ionizing at least a portion of said sample,a mass analyzer for mass-to-charge analyzing said ions, and a massanalyzer detector for detecting said mass-to-charge analyzed ions. 31.An apparatus according to claim 30, wherein said ion source comprises anElectrospray ion source.
 32. An apparatus according to claim 26 whereinsaid semipermeable membrane comprises an element selected from the groupconsisting of a cation exchange membrane, an anion exchange membrane, asize exclusion membrane, and single or multiple layered membranes. 33.An apparatus according to claim 26 wherein said detector comprises alight absorption detector.
 34. An apparatus according to claim 26wherein the pressure is regulated in said at least one membrane assemblysaid first and said second flow channels to control or minimize thepressure gradient across said semipermeable membrane configured in atleast one said membrane assembly.
 35. An apparatus according to claim 26wherein an electrode is positioned in said second flow channel of atleast one said membrane assembly.
 36. A method for analyzing chemicalspecies comprising: a. utilizing an apparatus comprising at least onemembrane assembly each comprising a semipermeable membrane, a first flowchannel having a first flow channel inlet and a first flow channeloutlet, a second flow channel having a second flow channel inlet and asecond flow channel outlet, said semipermeable membrane separating saidfirst and second flow channels; b. flowing a first and a second solutionflowing in said at least one first and second said flow channels,respectively; c. introducing sample species into a sample solutionflowing through said first flow channel; d. moving chemical speciesacross said at least one semipermeable membrane using concentrationsgradients of said species between said first and second solutions insaid at least one first and second flow channels respectively; e.detecting at least a portion of said sample species flowing from saidfirst solution flow outlet of at least one of said membrane assemblies37. A method according to claim 36 wherein an element selected from thegroup consisting of a mass spectrometer and a light detector is used todetect said chemical species.
 38. A method according to claim 36 whereina chemical species concentration in at least one second flow channel ishigher than the concentration of said species in said first solutionflow channel, effecting transfer of said chemical species through saidsemipermeable membrane.
 39. A method according to claim 36 wherein achemical species concentration in at least one second flow channel isvaried to control the exchange of said species through saidsemipermeable membrane from said second flow channel to said first flowchannel or in reverse.
 40. A method according to claim 36 wherein achemical or ionic species transfer through said in at least one secondflow channel is controlled by applying a voltage to an electrodepositioned in said second flow channel.
 41. A method according to claim36 wherein said chemical species comprises deuterated water, deuteratedhydronium ions or deuterium neutral or ion species to effect deuteriumexchange with said sample species.
 42. A method according to claim 36wherein an injector valve, liquid chromatography column or capillaryelectrophoresis column are utilized to introduce sample species intosaid first solution.
 43. A method for analyzing chemical speciescomprising: a. utilizing an apparatus comprising at least one membraneassembly each comprising a semipermeable membrane, a first flow channelhaving a first flow channel inlet and a first flow channel outlet, asecond flow channel having a second flow channel inlet and a second flowchannel outlet, said semipermeable membrane separating said first andsecond flow channels, and an electrode positioned in said second flowchannel; b. flowing a first and a second solution flowing in said atleast one first and second said flow channels, respectively; c.introducing sample species into a sample solution flowing through saidfirst flow channel; d. moving chemical species across said at least onesemipermeable membrane between said first and second solutions in saidat least one first and second flow channels respectively; e. detectingat least a portion of said sample species flowing from said firstsolution flow outlet of at least one of said membrane assemblies.
 44. Amethod according to claim 43 wherein an element from the groupconsisting of a mass spectrometer and a light detector is used to detectsaid chemical species.
 45. A method according to claim 43 wherein achemical species is transferred through said semipermeable membrane fromsaid first flow channel to said second flow channel by applying avoltage to said electrode positioned in said second flow channel.
 46. Amethod according to claim 43 wherein a chemical or ion species istransferred through said semipermeable membrane from said second flowchannel to said first flow channel by applying a voltage to saidelectrode positioned in said second flow channel.
 47. A method accordingto claim 43 wherein a chemical or ion species concentration in at leastone second flow channel is varied to control the exchange of saidspecies through said semipermeable membrane from said second flowchannel to said first flow channel or in reverse.
 48. A method accordingto claim 43 wherein said chemical species comprises deuterated water,deuterated hydronium ions or deuterium neutral or ion species to effectdeuterium exchange with said sample species.
 49. A method according toclaim 43 wherein an injector valve, liquid chromatography column orcapillary electrophoresis column are utilized to introduce samplespecies into said first solution.
 50. A method for analyzing chemicalspecies comprising: a. utilizing an apparatus comprising at least onemembrane assembly each comprising a semipermeable membrane, a first flowchannel having a first flow channel inlet and a first flow channeloutlet, a second flow channel having a second flow channel inlet and asecond flow channel outlet, said semipermeable membrane separating saidfirst and second flow channels, at least one said membrane assemblypositioned at the inlet of a liquid chromatography column and anelectrode positioned in said second flow channel; b. flowing a first anda second solution flowing in said at least one first and second saidflow channels, respectively; c. introducing sample species into a samplesolution flowing through said first flow channel; d. moving chemicalspecies across said at least one semipermeable membrane between saidfirst and second solutions in said at least one first and second flowchannels respectively; e. detecting at least a portion of said samplespecies flowing from said first solution flow outlet of at least one ofsaid membrane assemblies
 51. A method according to claim 50 wherein thepressure is regulated in said at least one membrane assembly said firstand said second flow channels to control or minimize the pressuregradient across said semipermeable membrane in at least one saidmembrane assembly.
 52. A method according to claim 50 wherein an elementselected from the group consisting of a mass spectrometer and a lightdetector is used to detect said chemical species.
 53. A method accordingto claim 50 wherein a chemical or ion species is transferred throughsaid semipermeable membrane from said first flow channel to said secondflow channel by applying a voltage to said electrode positioned in saidsecond flow channel.
 54. A method according to claim 50 wherein achemical or ion species is transferred through said semipermeablemembrane from said second flow channel to said first flow channel byapplying a voltage to said electrode positioned in said second flowchannel.
 55. A method according to claim 50 wherein a chemical speciesconcentration in at least one second flow channel is varied to controlthe exchange of said species through said semipermeable membrane fromsaid second flow channel to said first flow channel or in reverse.
 56. Amethod according to claim 50 wherein said transfer of said chemical orion species effects selective binding or release of said sample speciesin said chromatography column.
 57. A method according to claim 50wherein said liquid chromatography column comprises a packed reversephase, normal phase or ion exchange or an open capillary electrophoresiscolumn.